基因枪介导的籼稻遗传转化及外源基因在受体中的遗传研究

由来自水稻成熟胚的愈伤组织或由此而建立的悬浮细胞系作基因枪转化的靶材料,将质粒ｐＩＬＴＡＢ２２７（含ｈｐｈ基因和ｇｕｓ基因）导入籼稻,在Ｂａｓｍａｔｉ－１、青油粘、新山粘２９、胜优２号,明恢６３等品种获得了转基因植株。Ｔ０转化体ｇｕｓ基因组织化学染色和ＤＮＡ分子杂交（Ｓｏｕｔｈｅｒｎ ｂｌｏｔ）证实,ｇｕｓ和ｈｐｈ基因已整合到上述品种的基因组中。本实验仔细研究了ｈｐｈ基因在Ｂａｓｍａｔｉ－１的Ｔ１     

竹节花黄斑驳病毒启动了在棉花维管组织中是一个强启动子

用竹节花黄斑驳病毒（Ｃｏｍｍｅｌｉｎａ Ｙｅｌｌｏｗ Ｍｏｔｔｌｅ Ｖｉｒｕｓ,ＣｏＹＭＶ）启动子－ｇｕｓ嵌合基因和ＣａＭＶ３５Ｓ－ｇｕｓ嵌合基因通过根癌土壤杆菌（Ａｇｒｏｂａｃｔｅｒｉｕｍ ｔｕｍｅｆａｃｉｅｎｓ（Ｓｍｉｔｈ ｅｔ Ｔｏｗｎｓｅｎｄ）Ｃｏｎｎ）ＬＢＡ４４０４介导分别转入棉花（Ｇｏｓｓｙｐｉｕｍ ｈｉｒｓｕｔｕｍ Ｌ．ｃｖ．Ｊｉｎｇｍｉａｎ７,Ｊ７Ｇ）,诱导再生了棉花转基因植株。Ｇ     

玉米19kD醇溶贮藏蛋白基因启动子种子特异性表达控制区段的分析

为研究玉米（Ｚｅａｍａｙｓ　Ｌ．）１９ｋＤ醇溶贮藏蛋白（ｚｅｉｎ）基因启动子种子特异性表达的控制区段,将全长６９４ｂｐ的启动子进行５’端缺失,共得到６个缺失突变体,长度分别为４８８ｂｐ、３７８ｂｐ、３０２ｂｐ、１５２ｂｐ、１２４ｂｐ和８５ｂｐ。将６个片段分别与报告基因ｇｕｓ连接构建成表达载体ｐＤＧＢ系列,经土壤农杆菌（Ａｇｒｏｂａｃｔｅｒｉｕｍ）介导转化,引入烟草。ＧＵＳ活性检测证明,４８８ｂｐ启动子片段能促使ｇｕｓ基因在种子中特异表达。３７８ｂｐ、３０２ｂｐ、１５２ｂｐ和１２４ｂｐ片段启动子引导的ｇｕｓ基因在烟草根、叶柄、种子中均可表达。     

转基因植物转录后基因沉默的克服对策

人们对植物进行遗传转化的目的是让转基因在植物基因组中能够稳定整合并在当代及其子代中能够有效、稳定的表达 ,但是由于多种因素的影响 ,转基因在植物中的表达并不很理想。自从 1 986年Ｐｅｅｒｂｏｔｔｅ报道转基因烟草中转基因发生沉默以来 ,很多学者也都发现大量的转基因植株不能正常表达[1]。经过多年的研究 ,发现导致转基因沉默的机理有多种 ,根据其作用机理和水平的不同 ,人们通常把转基因沉默分为[2 ,3]:位置依赖性基因沉默 (Ｐｏｓｔｉｏｎ -ｄｅｐｅｎｄｅｎｔｇｅｎｅｓｉｌｅｎｃｉｎｇ ,ＰＤＧＳ)、转录水平的基因沉默 (Ｔｒ…     

利用Tn5gusA5对大豆慢生根瘤菌lrp基因的诱变

通过对重组质粒ｐＧＸＮ３００中的 ２．３ｋｂ ＥｃｏＲＩ片段测序分析发现,其上有一完整的ｌｒｐ基因和部分 ｐｕｔＡ基因,与 Ｋｉｎｇ ＮＤ等［１］报道的 Ｂ．ｊａｐｏｎｉｃｕｍ的ｌｒｐ基因ＤＮＡ序列有 ８８％同源性。利用 Ｔｎ５ ｇｕｓＡ５定位 诱变方法,对质粒ｐＧＸＮ３００进行插入诱变,得到２．３ｋｂ　ＥｃｏＲＩ片段上有Ｔｎ５ｇｕｓＡ５插入位点的质粒ｐＧＸＮ３００－ Ｔ３８,将ｐＧＸＮ３００－Ｔ３８转移到大豆馒生根瘤苗（Ｂ．ｊａｐｏｎｉｃｕｍ）ＧＸ２０１中,得到的ＧＸ２０１转移接合子与不相容 质粒ｐＰＨ１ＪＩ发生同源双交换。通过抗性及ｇｕｓＡ活性检测,筛选到一ｌｒｐ基因突变株。Ｓｏｕｔｈｅｍ杂交分析证 明这突变株的 Ｔｎ５ ｇｕｓＡ５插入确实是同源交换而不是转座产生,表明 Ｔｎ５ ｇｕｓＡ５ 诱变可以应用于大豆慢生根 瘤菌中的突变林筛选。     

植物转基因沉默及对策

转基因 (ｔｒａｎｓｇｅｎｅ)是指所有通过基因工程手段构建 ,导入受体生物细胞并稳定整合到该受体细胞基因组中的外源基因。人们对植物进行遗传转化的最终目的是让转基因在受体植物基因组中得到稳定整合并在当代及其子代中得到有效、稳定的表达 ,但是由于存在多种影响因子和限制因素 ,使得转基因在受体植物中的表达往往事与愿违。现实证明 ,转基因在受体植物中往往不能稳定表达 ,有时甚至完全不表达 ,出现了所谓的转基因沉默现象 (ｔｒａｎｓｇｅｎｅｓｉｌｅｎｃｉｎｇ)。转基因沉默并不等同于由于转基因在受体细胞中ＤＮＡ序列的歧变或因…     

表达barstar基因及bar基因的转基因油菜的研究

从细菌Ｂａｃｉｌｕｓａｍｙｌｏｌｉｑｕｅｆａｃｉｅｎｓ染色体ＤＮＡ中克隆了ｂａｒｎａｓｅ抑制剂ｂａｒｓｔａｒ的基因,构建了带有ＴＡ－２９基因５′调控区（－１３００－＋３）与ｂａｒｓｔａｒ基因编码区、ＣａＭＶ３５Ｓ启动子与除草剂抗性基因ｂａｒ两个表达框架的植物表达质粒ｐＢＢＳ。以“双低”油菜“５－４”的子叶柄为受体,通过农杆菌介导的遗传转化,获得了在含有１０ｍｇ／Ｌ卡那霉素和２０ｍｇ／ＬＰＰＴ的筛选培养基上再生的转基因植株。ＰＣＲ分析结果表明,ｂａｒｓｔａｒ基因已整合到油菜染色体上;Ｎｏｒｔｈｅｒｎｂｌｏｔ检测表明,ｂａｒｓｔａｒ基因及ｂａｒ基因在转基因植物中得到了正确的调控与表达。以转基因油菜“５－４”为父本授粉给表达ｂａｒｎａｓｅ基因的雄性不育植株,不育株能正常结实。     

基因沉默

基因沉默 (ｇｅｎｅｓｉｌｅｎｃｉｎｇ)是指生物体中特定基因由于种种原因不表达。一方面 ,基因沉默是遗传修饰生物 (ｇｅｎｅｔｉｃａｌｌｙｍｏｄｉｆｉｅｄｏｒｇａｎｉｓｍｓ)实用化和商品化的巨大障碍 ,另一方面 ,基因沉默是植物抗病毒的一个本能反应 ,为用抗病毒基因植物工程育种提供了具有较大潜在实用价值的策略———ＲＮＡ介导的病毒抗性 (ＲＮＡ ｍｅｄｉａｔｅｄｖｉｒｕｓｒｅｓｉｓｔａｎｃｅ ,ＲＭＶＲ) [1～ 3] 。基因沉默现象首先在转基因植物中发现 ,接着在线虫、真菌、昆虫、原生动物以及老鼠中陆续发现。大量的研究表…     

大肠杆菌海藻糖的代谢调控

海藻糖是一种重要的抗逆物质。大肠杆菌中ｏｔｓＢＡ操纵子编码的两种酶负责海藻糖合成。ｏｔｓＢＡ基因的表达受渗透压诱导和σ＾ｓ因子的调节。细胞的周质海藻糖酶（ｔｒｅＡ）将外源海藻藻分解成两个葡萄糖分子。尽管大肠杆菌中渗透压诱导合成的海藻糖并不能保护细胞抗干燥,我们将ｏｔｓＡ单个基因通过农杆菌转入烟草时,转基因株提高了耐盐和抗干燥特性,同时在转基因烟草提取物中检测到海藻糖,证明ｏｔｓＡ基因在烟草中表达并合成海藻糖。我们认为若将ｏｔｓＡ基因转入其它植物,可望改善这些植物的抗干旱、耐盐碱特性和延长采摘后的保鲜期。     

安全标记基因在转基因植物中的应用

转基因植物的抗性标记一直是转基因生物安全性争论的焦点,是限制转基因植物应用的瓶颈之一。筛选安全标记基因替代抗生素标记基因已成为解决转基因植物安全性和促进转基因植物应用的重要策略。综述了生物安全标记基因的产生背景、系统分类、筛选原理及不同起源的标记基因在植物基因工程中的应用和存在问题。选用植物内源标记基因已成为转基因植物安全标记基因研究的重要方向。     

Molecular and cellular evidence of chimaeric tissues in primary transgenics and elimination of chimaerism through improved selection protocols

Transgenic plants of strawberry cultivar Totem were developed by Agrobacterium-mediated transformation using a plasmid vector containing gus and nptII genes. Parallel experiments were carried out with and without repeated subculturing (iterative cultures) for generation of transgenic shoots on selection medium. The selection levels in the non-iterative pathway were kept constant, while in the iterative protocol, stepwise increase of selection pressure was applied at different stages of tissue growth. Rooted transgenic plants obtained via both protocols were outplanted in soil. Random leaf samples of greenhouse-grown transgenics were analysed for the presence of gus gene sequences by Southern hybridization as well as gus expression on leaf and petiole tissues by X-Gluc histological assay. Random leaf samples analysed from individual transgenic events developed under iterative culture were positive for the gus insert as verified by Southern analysis confirming the presence of transgenes and lack of chimaeras. Leaf samples of the transgenic events from the non-iterative protocol were either positive or negative on Southern analysis indicating the chimaeric nature of the transgenic plants. The absence of gus sequences in the transgenic plants grown under the non-iterative protocol reinforced the necessity of iterative cultures along with stepwise increase in selection levels for generating non-chimaeric transgenics in strawberry. The gus expression was highly variable, irrespective of the iterative or non-iterative protocol used for transformation. We conclude that strawberry is highly prone to develop chimaeric transgenics if derived from primary regenerants and that the iterative culture technique effectively converts chimaeras to pure line transgenic plants     

Biolistic-mediated genetic transformation of cowpea (<Emphasis Type="Italic">Vigna unguiculata</Emphasis>) and stable Mendelian inheritance of transgenes

We describe a novel system of exploiting the biolistic process to generate stable transgenic cowpea (Vigna unguiculata) plants. The system is based on combining the use of the herbicide imazapyr to select transformed meristematic cells after physical introduction of the mutated ahas gene (coding for a mutated acetohydroxyacid synthase, under control of the ahas 5' regulatory sequence) and a simple tissue culture protocol. The gus gene (under control of the act2 promoter) was used as a reporter gene. The transformation frequency (defined as the total number of putative transgenic plants divided by the total number of embryonic axes bombarded) was 0.90%. Southern analyses showed the presence of both ahas and gus expression cassettes in all primary transgenic plants, and demonstrated one to three integrated copies of the transgenes into the genome. The progenies (first and second generations) of all self-fertilized transgenic lines revealed the presence of the transgenes (gus and ahas) co-segregated in a Mendelian fashion. Western blot analysis revealed that the GUS protein expressed in the transgenic plants had the same mass and isoelectric point as the bacterial native protein. This is the first report of biolistic-mediated cowpea transformation in which fertile transgenic plants transferred the foreign genes to next generations following Mendelian laws.     

玉米Ubi－1启动子在可育转基因玉米植株中的表达活性

本工作将玉米泛素基因－1启动子（Ubi-1)与大肠杆菌β－葡萄糖苷酸酶基因（gus,uidA)的编码区融合,通过基因枪粒子轰击方法转化来自水成熟胚盾片组织的I－型愈伤组织,经PPT选择获得可育的玉米转基因植株,并采用组织化学方法分析了Ubi-1启动子驱动的gus基因在不同组织,细胞中的表达活性,发现gus基因在除花药壁以外的其它所试组织中均可以有效表达。Ubi：GUS在花粉,卵细胞中T1代转基因植株未成熟胚中的表达显示该启动子在植株发育的早期阶段即具有活性。对T0代转基因植株的花粉进行GUS组织化学染色,gus基因呈1：1分离,显示外源基因在转基因植株中以孟德尔方式遗传。同时发现,使用玉米本身的启动子Ubi-1可以降低外源基因在转基因玉米中的拷贝数,进而避免基因沉默现象的发生。目前已得到第二代转基因种子。     

Antisense inhibition of flavonoid biosynthesis in petunia anthers results in male sterility.

Inhibition of flower pigmentation in transgenic petunia plants was previously accomplished by expressing an antisense chalcone synthase (chs) gene under the control of the cauliflower mosaic virus (CaMV) 35S promoter. This chimeric gene was not effective in inhibiting pigmentation in anthers, presumably because the viral CaMV 35S promoter was insufficiently expressed in cell types of this organ in which the pigments are produced. Insertion of the anther box, a homologous sequence found in other genes expressed in anthers, resulted in a modified expression pattern driven by this promoter, as monitored by the beta-glucuronidase (gus) gene. In addition to the basic CaMV 35S expression pattern in anthers, GUS activity was observed in tapetum cells when the modified promoter was fused to the gus gene. This promoter construct was subsequently used to drive an antisense chs gene in transgenic petunia, which led to the inhibition of pigment synthesis in anthers of five of 35 transformants. Transgenic plants with white anthers were male sterile due to an arrest in male gametophyte development. This finding indicated that flavonoids play an essential role in male gametophyte development.     

Competence of oat ( Avena sativa L.) shoot apical meristems for integrative transformation,inherited expression,and osmotic tolerance of transgenic lines containing hva1

Three oat ( Avena sativa L.) cultivars have been successfully transformed using an efficient and reproducible in vitro culture system for differentiation of multiple shoots from shoot apical meristems. The transformation was performed using microprojectile bombardment with two plasmids (pBY520 and pAct1-D) containing linked ( hva1-bar) and non-linked ( gus) genes. The hva1 and bar genes cointegrated with a frequency of 100% as expected, and 61.6% of the transgenic plants carried all three genes. Molecular and biochemical analyses in R0, R1 and R2 progenies confirmed stable integration and expression of all transgenes. Localization of the GUS protein in R0 and R1 plants revealed that high-expression of gus occurred in vascular tissues and in the pollen grains of mature flowers. The constitutive expression of HVA1 protein was observed at all developmental stages of transgenic plants, and was particularly stronger during the early seedling stages. R2 progeny of five independent transgenic lines was tested in vitro for tolerance to osmotic (salt and mannitol) stresses. As compared to non-transgenic control plants, transgenic plants maintained a higher growth and showed significantly ( P < 0.05) increased tolerance to stress conditions. Less than 10% of transgenic plants showed symptoms of wilting or death of leaves and, when these symptoms present were delayed in transgenic plants as compared to 80% of non-transgenic plants, either wilted or died. These symptoms confirmed the increased in vitro tolerance in hva1-expressing transgenic plants to non-transgenic plants, providing strong evidence that the HVA1 protein may play an important role in the protection of oats against salinity and possible water-deficiency stress conditions.     

The Competence of Maize Shoot Meristems for Integrative Transformation and Inherited Expression of Transgenes

We have developed a novel and reproducible system for recovery of fertile transgenic maize (Zea mays L.) plants. The transformation was performed using microprojectile bombardment of cultured shoot apices of maize with a plasmid carrying two linked genes, the Streptomyces hygroscopicus phosphinothricin acetyltransferase gene (bar) and the potato proteinase inhibitor II gene, either alone or in combination with another plasmid containing the 5[prime] region of the rice actin 1 gene fused to the Escherichia coli [beta]-glucuronidase gene (gus). Bombarded shoot apices were subsequently multiplied and selected under 3 to 5 mg/L glufosinate ammonium. Co-transformation frequency was 100% (146/146) for linked genes and 80% (41/51) for unlinked genes. Co-expression frequency of the bar and gus genes was 57% (29/51). The co-integration, co-inheritance, and co-expression of bar, the potato proteinase inhibitor II gene, and gus in transgenic R0, R1, and R2 plants were confirmed. Localized expression of the actin 1-GUS protein in the R0 and R1 plants was extensively analyzed by histochemical and fluorometric assays.     

Functional analysis of the ver gene using antisense transgenic wheat

The function of ver203 , a gene related to vernalization in winter wheat, was investigated by expression of a complementary DNA as an antisense RNA in transgenic plants. A verc203:gus fusion‐expression plasmid was constructed in pBI221, which contains a CaMV (cauliflower mosaic virus) 35S‐promoter, a gus gene and a nos terminator. The construct was then introduced into the plant by the pollen‐tube pathway. The results showed that heading was strongly inhibited in 6 of 326 vernalized antisense transgenic winter wheat plants, until both the vernalized control winter wheat and sense transgenic plants ripened. The hybridization analysis of DNA, amplification of the insert DNA sequences with PCR, northern blot analysis with double‐ and single‐stranded probes, and detection of GUS activity by X‐gluc assay gave strong positive results. This suggests that the VER203 protein plays an important role in controlling heading and flower development in winter wheat.     

Genetic transformation of potato with nptII-gus marker genes enhances foliage consumption by Colorado potato beetle larvae

Little is known about the effect of transgenic plants containing commonly used marker genes, such as aph(3)II (nptII encoding neomycinphosphotransferase) and uidA (gus encoding -glucuronidase) on insect feeding behaviour. We report here, for the first time, that transgenic potato plants containing only nptII and gus marker genes enhance foliage consumption by the Colorado potato beetle (CPB, Leptinotarsa decemlineata S.). Transformation of potato cultivar Désirée was performed with Agrobacterium tumefaciens. Internode explants were inoculated with different strains of bacteria, carrying either nptII-gus or nptII alone. A total of 180 transgenic and untransformed control plants were grown in the greenhouse for the analysis of food consumption by CPB. For each transformed and untransformed line tested, four bioassays were conducted each consisting of 10 second-instar larvae feeding independently on a 2 cm diameter leaf disc for 20 h. Our data show up to 50% increase of mean foliage consumption on plants transformed with the nptII-gus construct, indicating that transgenic plants containing these marker genes can affect the feeding behaviour of the insects. These results were obtained from the primary regenerants (R₀ lines) as well as from tuber-derived plants (R₁ lines). Further tests with transgenic plants containing the nptII marker gene only, showed no significant difference in feeding when compared to untransformed control plants, allowing us to rule out a direct effect of this marker gene on foliage consumption by the insect larvae. It is suggested that gus protein is involved in the increase of foliage consumption by CPB.     

The Commelina Yellow Mottle Virus Promoter Is a Strong Promoter in Vascular Tissue of Transgenic Gossypium hirsutum Plants

Explants of cotton (Gossypium hirsutum L. cv. Jingmian 7) were transformed with Agrobacterium tumefaciens (Smith et Townsend ) Conn LBA4404 harboring an expression cassette composed of CoYMV (Commelina Yellow Mottle Virus) promoter-gus-nos terminator on the plant expression vector pBcopd2. Transgenic plants were regenerated and selected on a medium containing kanamycin. GUS (β-glucuronidase) activity assays and Southern blot analysis confirmed that the chimerical gus gene was integrated into and expressed in the regenerated cotton plants. Plant expression vector pBI121 was also transferred into the same cotton variety and the regenerated transgenic plants were used as a positive control in GUS activity analysis. Evidences from histochemical analysis of GUS activity demonstrated that under the control of a 597 bp CoYMV promoter the gus gene was highly expressed in the vascular tissues of leaves, petioles, stems, roots, hypocotyls, bracteal leaves and most of the flower parts while GUS activity could not be detected in stigma, anther sac and developing cotton fibers of the transgenic cotton plants. GUS specific activity in various organs and tissues from transgenic cotton lines was determined and the results indicated that the CoYMV promoter-gus activities were at the same level or higher than that of CaMV 35S promoter-gus in leaf veins and roots where the vascular tissues occupy a relatively larger part of the organs, but in other organs like leaves, cotyledons and hypocotyls where the vascular tissues occupy a smaller part of the organs the CoYMV promoter-gus activity was only 1/3-1/5 of the CaMV 35S promoter-gus activity. The GUS activity ratio between veins and leaves was averaged 0.5 for 35S-GUS plants and about 2.0 for CoYMV promoter-gus transgenic plants. These results further demonstrated the vascular specific property of the promoter in transgenic cotton plants. An increasing trend of GUS activity in leaf vascular tissues of transgenic cotton plants developing from young to older was observed.